Development of a Multiplex PCR Assay for Detection of <i>Pseudomonas fluorescens</i> with Biofilm Formation Ability

Xu, Yu Quan; Chen, Wanyi; You, Chunping; Liu, Zhenmin

doi:10.1111/1750-3841.13845

Cited by 10 publications

(3 citation statements)

References 26 publications

(42 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For pseudomonads, two non-quantitative multiplex PCR approaches have been performed for different Pseudomonas species in meat (Ercolini et al 2007) and of P. fluorescens strains with a biofilm-forming ability (Xu et al 2017). However, until now, no qPCR assay has been developed for the simultaneous quantification of various milk-spoiling Pseudomonas species in Fig.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

Maier

Hofmann

Huptas

et al. 2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85–97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103–107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. Key points • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential

show abstract

Section: Discussionmentioning

confidence: 99%

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

Maier

Hofmann

Huptas

et al. 2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…This approach was successfully used in meat and dairy processing environment for the isolation of P. fluorescens oligotypes (Stellato et al 2017). Another study reported the use of multiplex PCR for the detection of P. fluorescens showing the ability of biofilm formation with a detection limit of target strain to 10 2 cfu/ml (Xu et al 2017).…”

Section: Enzymes Of P Fluorescensmentioning

confidence: 99%

Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection

et al. 2019

View full text Add to dashboard Cite

Purpose This review focuses on the spoilage strategies used by the Pseudomonas fluorescens, and in addition, it also discusses various diagnostic approaches used for its identification in food items. Some challenges faced and advances in the detection of P. fluorescens and also discussed in this review. Methods An extensive literature search was performed with published work and data was analyzed in detail to meet the requirements of the objectives. Results P. fluorescens are unicellular rods, with long straight or curved axis, but not helical, motility by one or more polar flagella, Gram-negative, non-spores former, stalks, or sheaths. P. fluorescens is represented by seven biotypes denoted by the letters A, B, C, D, E, F, and G. The microbe shows wide choice of growth temperature and causes contamination and spoilage in ordinary and refrigerated food items by its enzymes and pigment production. The biofilm formation by P. fluorescens poses another serious threat to the food industries. Conclusion Molecular identification of P. fluorescens is generally done by 16S rRNA, intergenic spacer (ITS1) utilizing traditional polymerase chain reactions (PCR). Nowadays, qPCR and multiplex PCR are largely utilized in identification of P. fluorescens based on AprX gene (extracellular caseinolytic metalloprotease) in the milk and meat spoilage strains. The available methods still show some disadvantages with accuracy and specificity of detection. Rapid detection of P. fluorescens in food samples is the need of hour to improve the detection efficiency.

show abstract

“…To reduce the frequency of contamination and to eliminate the niche populations for these organisms in food production and processing systems, different identification and tracking methods have been developed (Martin et al, 2011). However, the high genetic and genomic diversity within the P. fluorescens complex (Garrido-Sanz et al, 2016) found in cheese samples requires running different phenotypic tests, such as API 20 NE (Martin et al, 2011), MALDI-TOF (Hrabák et al, 2013), and distinct genotypic techniques, such as PCR (Scarpellini et al, 2004;Xu et al, 2017), 16S rRNA sequencing (Caputo et al, 2015;Cenci-Goga et al, 2014;Chierici et al, 2016;Garrido-Sanz et al, 2016), pulsed-field gel electrophoresis (PFGE; Martin et al, 2011;Carrascosa et al, 2015), and MLSA (Andreani et al, 2015a,b;Gomila et al, 2015;Chierici, 2016;Garrido-Sanz et al, 2016), to avoid making mistakes in their correct identification.…”

Section: Introductionmentioning

confidence: 99%

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese

Carrascosa

Martínez

Velázquez

et al. 2021

Journal of Dairy Science

View full text Add to dashboard Cite

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.

show abstract

Development of a Multiplex PCR Assay for Detection of Pseudomonas fluorescens with Biofilm Formation Ability

Abstract: A mPCR assay targeting adnA and fliC genes showed rapid and reliable detection of P. fluorescens with biofilm formation ability, which could be useful to detect this contamination in milk samples.

Cited by 10 publications

References 26 publications

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese

Contact Info

Product

Resources

About